Alpha-Emitting constructs and uses thereof

ABSTRACT

The present invention discloses alpha particle emitting, radioactive constructs capable of killing large tumors (&gt;1 mm in diameter), or other cells involved in human or animal diseases such as virus infected cells, autoimmune cells, or other pathological cells, including normal cells, that are targets for destruction, to achieve a therapeutic result. The alpha-emitting constructs have high specific activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of pending nonprovisional applicationU.S. Ser. No. 09/721,864, filed Nov. 24, 2000, which claims benefit ofpriority under 35 U.S.C. §120 of international applicationPCT/US1999/11673, filed May 26, 1999, now abandoned, which claimsbenefit of priority under 35 U.S.C. §119(e) of provisional applicationU.S. Ser. No. 60/086,772, filed May 26, 1998, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field ofradioimmunotherapy. More specifically, the present invention relates toalpha-emitting constructs with high specific activity and their uses tokill large tumors or other cells involved in human disease states.

2. Description of the Related Art

In radiolabeled antibody therapy, an antibody/radionuclide combinationthat has been optimized for bulky disease is not optimal for targetingminimal disease (1). Radionuclides that emit long-range beta particles,for example, are generally considered appropriate for targeting bulkydisease because their range compensates for the non-uniform antibodydistribution that is typical of gross disease. These radionuclides,however, are inappropriate for targeting single cells (2-3).

Antibody forms such as fragments that penetrate solid tumor more rapidlydo so at the expense of affinity. In targeting smaller, more penetrableclusters, such agents are only left with the disadvantage of reducedaffinity. This is in contrast to chemotherapy wherein a greatereffectiveness against bulky disease is also applicable to minimaldisease. Radiolabeled antibody therapy is fundamentally different fromchemotherapy in its mechanism of action. Although well-justified forchemotherapy, the “solid-tumor hurdle” is not appropriate forradioimmunotherapy (4).

To target bulky disease, intravenously administered antibody mustextravasate, diffuse across an interstitial fluid space and thendistribute throughout antigen positive cells (FIG. 1A). Each of thesesteps is associated with a barrier to delivery (5-11). By targetinghematologically distributed single tumor cells or tumor cell clusters,the barriers to antibody delivery are diminished (FIG. 1B). The apparenttherapeutic dependence on cluster size and tumor burden is consistentwith modeling analyses and experimental observations of antibodypenetration (12).

In targeting disseminated tumor cells or micrometastases, each tumorcell must express the antigen. This seemingly severe requirement may becountered by taking advantage of the unique aspects of micrometastatictargeting. Intravenously administered antibody will not be rapidlyaccessible to potentially cross-reactive cells on the epithelial side ofthe vasculature. It is possible, therefore, to relax the requirements ofantibody specificity when targeting rapidly accessible, hematologicallydistributed disease by using shorter-lived radionuclides which will havedecayed before antibody extravasation. By relaxing the requirement forspecificity, antigens that have a higher and more uniform expression ontumor cells may be selected (13).

Bismuth-213 or 212 conjugated alpha-emitting IgG ligands have beenproposed to be useful in humans in killing single cells only. Theseligands have not thought to be useful in killing solid tumors or smallmicrometastatic collections of cells only. These single cells orclusters of cells are found in the blood, bone marrow, lymph nodes,liver, and spleen or in regional collections as small metastaticdeposits such as in the cerebrospinal fluid, ascites or pleural fluidsof patients with leukemia and other cancers (“For Bi-213, no specifickilling was observed, which is an indication for the limitedapplicability of this radionuclide in the treatment of solid tumors.”;14-20). This universally held belief of the application of alphaparticle emitters to single cells was based on the short path length ofthe alpha particles (<100 micrometers), equal to about 2-4 celldiameters and the short half-life of the nuclides (<1 hr). Because thealpha particle emitter decays largely within 3-4 hours, and the time foran IgG to diffuse into a large tumor is on the order of days, there wasthought to be little possibility that an IgG carrying Bi-213 or Bi-212could penetrate beyond 1 or 2 cells to achieve cell kill. Hence, onlysingle cells in the blood, marrow, liver or spleen would be reasonabletargets. It is for that reason that the initial studies have focused onleukemias, peritoneal metastases, cancerous meningitis in thecerebrospinal fluid, or micromestatic deposits in the bone marrow.

Strategies to use small quantities, 5-20 mCi, of Bi-213 on labeledligands have been proposed to kill individual cells such as cancercells. This strategy involves the use of single doses of Bi-213 labeledantibody or other ligands. However, these methods alone do not enablethe use of alpha particle emitting constructs because they fail to takeinto account the necessity for high specific activity ligands in orderfor specific cell kill to occur. This necessity arises from theparticular nature of the alpha particle emission, i.e., high linearenergy transfer and extremely short range, which does not exist for thebeta or gamma emissions that have previously been used therapeuticallyin humans. As a consequence, whereas a beta emitting therapeuticantibody which kills in a field of radiation may be effective at anynumber of specific activities, an alpha-emitting antibody will only beeffective if a minimum of one atom can be delivered to each cell,resulting in at least 1 alpha track through the cell.

The prior art is deficient, first, in understanding the importance ofand requirement for the high specific activity in the process of cellkill with an alpha particle, and second, in understanding how one mightkill tumors with more than a small number of cells. Therefore, the priorart is deficient in the lack of effective means of killing large tumors(>1 mm in diameter) or other cells involved in human or animal diseasesusing the high specific alpha-emitting constructs. The present inventionfulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention discloses Bismuth-213 labeled, alpha particleemitting, radioactive constructs capable of killing large tumors, >1 mmin diameter, or other cells involved in human or animal diseases such asvirus infected cells, autoimmune cells, or other pathological cells,including normal cells, that are targets for destruction, to achieve atherapeutic result. Methods to kill large tumors, previously thought tobe impossible due to the short range of the alpha particles, aredescribed and documented in vitro and in vivo against a human cancermodel. The necessity for high specific activity constructs (the numberof isotope atoms per ligand molecule) to enable therapeutic results isshown. The necessity for even higher specific activity to achieve “cure”of a cancer, also known as “tumor control probability (TCP)” of one, isalso described. These concepts have not been disclosed before in theart, or have been taught away from in the art, and hence, areunexpected.

The present invention also investigates possible therapeutic activitiesof a longer-lived alpha-emitting nuclide (Ac-225) with multiplealpha-emitting daughters. New chelates containing Ac-225 and itsdaughters are examined.

Drugs based on alpha-emitting particles can be prepared and administeredsafely and repeatedly without extramedullary toxicity. The drug madefrom Bi-213 labeled constructs displayed pharmacokinetics consistentwith rapid, specific, and stable targeting only to appropriate cancercell sites. Significant anti-leukemic activity was seen at the lowestlevel. Drugs made from Ac-225 labeled constructs are shown to be morepotent, as much as 1000 fold more on a mCi basis, than Bi-213constructs.

In one embodiment of the present invention, there is provided a methodof killing a large tumor comprising the step of administering analpha-emitting construct to the tumor repeatedly. Specifically, thetumor is larger than 1 mm in diameter. Representative examples ofalpha-emitting constructs include an antibody, a fragment, a cytokine, areceptor ligand and a ligand of any other kind. Preferably, thealpha-emitting construct is labeled by bismuth-213, bismuth-212,actinium-225, radium-223, lead-212, terbium-149, fermium-155 orastatine-211. The construct should have a high specific activity of fromabout 0.05 mCi/mg to about 100 mCi/mg. Further, the alpha-emittingconstruct is administered by repeated dosing in a range of from about0.1 mg/m² to about 10 mg/m².

In another embodiment of the present invention, there is provided amethod of killing a non-malignant cell in a human comprising the step ofadministering an alpha-emitting construct to the cell. Representativeexamples of alpha-emitting constructs include an antibody, a fragment, acytokine, a receptor ligand and a ligand of any other kind. Preferably,the alpha-emitting construct is labeled by Bismuth-213, Bismuth-212,actinium-225, radium-223, lead-212, terbium-149, fermium-155 orastatine-211. More preferably, the construct has a high specificactivity of from about 0.05 mCi/mg to about 100 mCi/mg, depending on theparticular isotope, and is administered in an effective dosage of fromabout 0.1 mg/m² to about 50 mg/m². Preferably, the non-malignant cell isselected from the group consisting of a virus-infected cell, anautoimmune cell, a lymphoid cell, a normal bone marrow cell and anovergrown normal cell. Representative examples of diseases that may betreated using this method include non-neoplastic disease, viralinfection, autoimmune disease, prostatic hypertrophy, coronary diseaseand other vascular occlusive disease.

In another embodiment of the present invention, there is provided amethod of killing a tumor by targeting antigens in the tumor vasculatureof an individual in need of such of such treatment, comprising the stepof: administering to said individual a pharmacologically effective doseof a construct comprising an alpha-emitting isotope effective to inhibitthe function of said tumor vasculature.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIGS. 1A-1B show barriers to antibody targeting of tumor cells. FIG. 1Ashows solid-tumor targeting. Antibody (Y) must first extravasate fromthe capillary (cylinder), then diffuse across an interstitial fluidpressure gradient (arrows) to reach the tumor cells (spheres). FIG. 1Bshows targeting hematologically distributed micrometastases. Antibodyextravasation and diffusion across the interstitial fluid are notnecessary. Single tumor cells are directly accessible.

FIG. 2 shows killing of large spheroids as one would “peal an onion.”Both spheroid sequences show images obtained after an overnightincubation with Bi-213-labeled antibody. The top sequence shows thattreatment with the PSMA specific antibody kills a layer of cells arounda core that does not grow further. Additional rounds of treatment willthen eliminate the core. In contrast, the spheroids in the lowersequence were treated with a control nonspecific construct and continueto grow.

FIGS. 3A-3B show the potency of (Bi-213)CHX-A-DTPA-HuM195 and(Bi-212)CHX-A-DTPA-HuM195 at killing leukemia cells in vitro.Cytotoxicity was measured using HL60 cells (CD33+) (dotted lines) andRAJI cells (CD33−) (solid lines) using specific activities ranging from0.2 mCi/mg to 30 mCi/mg. 2×105 cells in 100 ml were placed in 96 wellplates.

Bismuth labeled antibody was added in the wells in serial dilution sothat final concentrations in the wells were in a range of 0.02 to 20mCi/ml. The plates were incubated 24 h at 37° C. in 5% CO2. Viabilitywas determined by 3H-thymidine incorporation, and is plotted againstspecific activity. FIG. 3A shows cytotoxicity with(Bi-212)CHX-A-DTPA-HuM195 as a function of specific activity and dose:0.2 mCi/mg (HL60: dotted lines, open symbols and RAJI: solid lines,closed symbols) 0.2 mCi/mg (crosses), 10 mCi/mg (circles), 20 mCi/mg(diamonds) and 30 mCi/mg (squares). FIG. 3B shows cytotoxicity of(Bi-213)CHX-A-DTPA-HuM195 as a function of specific activity and dose.RAJI: (closed symbols) 2 mCi/mg (circles) and 8 mCi/mg (triangles);HL60: (open symbols) 1 mCi/mg (triangles), 2 mCi/mg (circles), 4 mCi/mg(diamonds) and 8 mCi/mg (squares).

FIGS. 4A-4B shows HL60 cell viability as a function of the calculatedaverage number of Bi-213 atoms (FIG. 4A) and Bi-212 atoms (FIG. 4B)bound on the cell surface. The line represents a linear best fit for thecytotoxicity data points taken from bismuth labelings that yielded aspecific activity of about 10 mCi/mg. This specific activity is in theregion of highly selective killing of HL60 cells. Curves of similarslope can be generated from cytotoxicity data at other high specificactivities (not shown).

FIG. 5 shows the potency of Bi-213-J591 against LnCaP cell viability asa function of specific activity. As the specific activity is lowered,the ability of the same concentration of isotope to kill the targetcells is reduced dramatically.

FIG. 6 shows Kaplan-Meier survival plot of fraction of mice survivingtumor-free vs. time for LNCaP xenografted mice treated with Bi-213-J591,Bi-213-HuM195, and an untreated growth control.

FIG. 7 shows mean PSA values at day 30 for LNCaP xenografted micetreated with Bi-213-J591, Bi-213-HuM195, unlabeled J591, and anuntreated growth control.

FIG. 8 shows Kaplan-Meier survival plot of fraction of mice survivingtumor-free vs. time for LNCaP xenografted mice treated with Ac-225labeled anti-PSMA IgG (J591), irrelevant Ac-225 labeled IgG, unlabeledPSMA IgG, and untreated growth controls.

DETAILED DESCRIPTION OF THE INVENTION

Strategies to use small quantities (5-20 mCi) of Bi-213 on labeledligands have been proposed to kill individual cells such as cancercells. This strategy involves the use of single doses of Bi-213 labeledantibody or other ligands. However, these methods alone do not enablethe use of alpha particle emitting constructs because they fail to takeinto account the necessity for high specific activity ligands in orderfor specific cell kill to occur. This necessity arises from theparticular nature of the alpha particle emission, that is, high linearenergy transfer and extremely short range, which does not exist for thebeta or gamma emissions that have been used therapeutically in humans.As a consequence, whereas a beta emitting therapeutic antibody thatkills in a field of radiation may be effective at any number of specificactivities, an alpha-emitting antibody will only be effective if aminimum of one atom can be delivered to each cell. In order to calculatethe specific activity required (atoms of isotope per ligand molecule (ormCi per mg)), an understanding of (1) the target site number and (2)modulation and pharmacology of the ligand is required. Site number alonemay differ over 1000 fold between different systems. As an example ofthe wide ranges of target site and ligand characteristics see Table 1:

TABLE 1 Target No. of sites per cell Ligand Affinity Growth factorreceptor 1000 0.1 nM (growth factor) on myeloid cell CD33 on leukemiacell 10,000   1 nM (M195 IgG) GD3 on melanoma cell 1,000,000  10 nM (R24IgG3)

The minimum requirements to achieve reliable cell killing depend on: (1)the number of receptor targets (binding sites) on the target cell; (2)the stability of the ligand at the site once targeted; (3) the rapidityby which the ligand reaches the target site; (4) the affinity of theligand for its target; and (5) the total number of target sites or nonspecific binding sites in the host (patient). With approximations ofeach of these features it is possible to estimate the specific activitynecessary to make an effective agent in each therapeutic application.For example, if one Bi-213 disintegration, with a T½ of 46 min, isnecessary to achieve single cell kill, and the radioactive ligand isstable at the cell for at least 3 hours, and 50% of the decays are inthe wrong direction and expend their energy outside of the cell, and itrequires 23 minutes for the ligand to reach the cell after injection and23 minutes to prepare the manufactured dose and administer it into thepatient, and there are 10,000 binding sites per cell, then a minimum of1 out of every 2500 ligands must be labeled with a Bi-213 atom at thetime of the end of the reaction to produce the radioligand. That is, attime=0 there is 1 Bi atom for each 2500 IgG; at time=46 minutes there is1 atom for each 5000 IgG. Since there are 10,000 possible sites percell, then 2 atoms will reach the cell and over 3 hours one will decaywith an alpha into the cell and one, away from the cell, on average.

This is a rough estimate of the conditions for treating a leukemia withBi-213 labeled HuM195 IgG (see example of its successful use in humansbelow). Thus, a minimum specific activity of about 10 mCi/mg isnecessary. Since there is a range of 1-50E10 possible target leukemiacells (i.e. 0.1-5 E15 binding sites at 10,000 sites per cell) dependingon the stage of disease, and about 50% of the antibody ultimately bindsthe target cell due to its affinity, a dose range of 0.05-2.50 mg ofantibody is needed to saturate all the available binding sites anddeliver an adequate dose. In contrast, for a ligand with similarpharmacology and preparation time, but 10 fold less sites, e.g. atypical cell surface receptor, a specific activity of nearly 1 atom per250 ligands is required. If this were an IgG, then approximately 100 mCiper mg is necessary. Failure to use this level of specific activitywould result in most cells receiving no radioactive atoms and hence aninability to adequately kill the target as compared to normal tissues.Thus, a minimum adequate specific activity of the radioactive constructis an integral characteristic of its description. Without this featuredescribed it is not possible for someone skilled in the art to prepare auseful dose. Note that this concept is not necessary for use of an“immunotoxin” because in this case each IgG is labeled with a toxinmolecule; moreover, the concept is not necessary for use with ligandslabeled with beta emitters, I-131 or Y-90, etc., since these isotopeskill in a large field rather than at the individual cell level.Therefore this concept is unique and not previously described forradioimmunotherapy.

A second important concept is the relationship between the specificactivity and the tumor control probability. The radiobiological issuesassociated with radioimmunotherapy of micrometastases are critical toproper treatment design and to the interpretation of clinical results.The initial criterion for effectiveness is much more difficult to meetin targeting micrometastases than it is in the treatment of measurable,bulky disease. In bulky disease, the initial criterion for effectivenessis the attainment of a complete response. If one assumes that the limitof detectability in a patient is 1 gram or 10⁹ cells, a completeresponse of a 100 gram lesion requires two logs of cell kill. Onceadequate cell kill has been achieved, remission depends on the durationof the eradication. Durable remissions are much more difficult toachieve. In the adjuvant treatment of micrometastases, the remissionduration will be the primary measure of effectiveness. This will dependupon the fraction of cells surviving after radioimmunotherapy and alsoon the time required for the population of cells to double when cellloss is negligible, i.e., the potential doubling time. The potentialdoubling time of most tumor cells ranges from 2 to 15 days (21). If theinitial treatment yields a complete remission by reducing the tumor massfrom 100 gm. to 0.1 gm, which is 3 logs of cell kill, and if thepotential doubling time of the tumor cells is 15 days, then in about 2months the tumor will have re-grown to 1 gram and the first evidence ofa recurrence will be apparent. If adjuvant treatment yields another 3logs of cell kill, following the initial 3 logs of kill, then 10⁵ tumorcells will remain. Assuming, a potential doubling time of 15 days,approximately 7 months will be required before the tumor becomesdetectable. If only a single cell survives after adjuvant treatment, arecurrence may be expected in 15 months.

To achieve a cure, i.e., 5-year disease-free survival, the probabilityof killing all tumor cells must approach 1. This probability isequivalent to the tumor control probability (TCP) and may be calculatedfrom: TCP=e^(−n*SF) where n is the initial number of tumor cells, SF isthe surviving fraction after treatment and e is Euler's number (2.71828. . . ). If, for example, following surgery or radiotherapy 10⁵ tumorcells remain and these are reduced to a single cell (n=10⁵; SF=10⁻⁵)then the probability of tumor control is 0.37. An additional log of cellkill increases the control probability to 90%; 99% probability isachieved after 7 logs of cell kill. Four logs of cell kill, which wouldreduce the tumor cell number from 10⁵ to 10 cells, only yields a tumorcontrol probability of 0.005%. In clinical terms, 37% of patients with10⁵ tumor cells would achieve a 5-year disease-free survival iftreatment potency was such that 10⁵ tumor cells could be reduced to 1tumor cell. If the treatment were potent enough to reduce 10⁷ cells to asingle cell, 99% of patients with 10⁵ cells would be disease-free after5 years. If, in the same patient population, treatment reduced the totalnumber of cells to 10 from 10⁵, only 5 in every 100,000 patients wouldbe cured.

Although the analysis presented above is a simplification that ignores alarge number of factors that are operative in determining the time torecurrence and the probability of cure, the exercises highlight thefundamental differences between the criterion used for evaluating anagent against measurable versus micrometastatic disease and betweenremission and a durable remission/“cure”. As is evident from theanalysis, the number of tumor cells remaining in the patient aftertreatment of the primary or observable metastases will be a criticaldeterminant of efficacy.

All previously described alpha-emitting constructs were of such lowspecific activity that construction of ligand to their specificationswould have yielded ineffective agents or agents unable to induce cureswhen injected into humans. Moreover, the consequences of this lowspecific activity were unrecognized until the work described herebecause the field had relied exclusively on the use of beta and gammaemitters, and because no one had ever successfully scaled up targetedalpha emitters for human use.

Besides Bi-213, a long-lived (t_(1/2)=10 days) alpha-emittingradionuclide Ac-225 is also investigated. Ac-225, with 3 alpha-emittingdaughters, should be far more potent than the short-lived (t_(1/2)=46min.) alpha-emitting Bi-213 in killing individual cells, if it retainedin or on the cell, and multicellular spheroids, if it can penetrate intothe spheroid over time. These features of Ac-225 may allow for alphatherapy of solid tumors; Bi-213 is unlikely to be useful for treatingsolid tumors unless multiple doses of drug can be used to slowly “peelaway” layers of tumor cells. The respective pros and cons to usingAc-225 and Bi-213 are shown in Table 2.

TABLE 2 Pros and cons of Ac-225 vs. Bi-213 use clinically Pro: Ac-225Con: Ac-225 Pro: Bi-213 Con: Bi-213 Long t_(1/2) = 10 days In vivostability of Short t_(1/2) = 46 min. Targeting must be allows time totarget chelate is means no long term rapid and penetrate tumor unknownresidual activity Daughters may yield Daughters may leak No long livedSingle alpha more alpha particles from target and kill daughtersemission and improve therapy nonspecifically Active agent might beActive agent must formulated and be prepared on site dispensed from afor rapid use in clinic central pharmacy Microcurie levels of activity(relative to Bi- 213) required

Using IgG-chelate constructs of relevant and control mAbs labeled withAc-225, the potency and specificity of Ac-225 labeled constructs areinvestigated in killing tumor cells and spheroids in vitro. The role ofthe constructs in cellular internalization and catabolism, and retentionof the nuclide in the cells are also evaluated.

It is hypothesized that generation of alpha-particles in vivo, at thetarget site, by targeting a long-lived isotope that decays via 4 alphas,which may be bound and retained within the target cell by modulationinto a cytoplasmic compartment, will yield a drug that is even morepotent, i.e., as much as 1000 fold more on a mCi basis, than Bi-213constructs. Moreover, the long half-life will allow targeting of solidtumors and larger micrometastatic lesions than are possible with theshort-lived Bi-213. Possible target systems under study include thebreast and prostate models. Total doses of less than 1 mCi areenvisioned.

In the present invention, high specific activity alpha-emittingconstructs are disclosed. Further, methods of killing large tumors orother cells involved in human or animal diseases using thealpha-emitting constructs are also disclosed.

The present invention is directed to a method of killing a large tumorcomprising the step of administering multiple doses of an alpha-emittingconstruct to the tumor. Specifically, the tumor is larger than 1 mm indiameter. Preferably, the alpha-emitting construct comprises anantibody, a fragment, a cytokine, a receptor ligand and other suchligands. Representative examples of alpha-emitting isotopes includeBismuth-213, Bismuth-212, actinium-225, radium-223, lead-212,terbium-149, fermium-155 and astatine-211. Generally, the construct hasa high specific activity of from about 0.1 mCi/mg to about 50 mCi/mg.That is, the construct is administered in a dose adequate to deliver aminimum of 1 atom per cell. Preferably, the alpha-emitting construct isadministered by repeated dosing in a range of from about 0.1 mg/m² toabout 10 mg/m².

The present invention is also directed to a method of killing anon-malignant cell involved in a human or an animal disease comprisingthe step of administering an alpha-emitting construct to the cell.Representative examples of alpha-emitting construct include antibodiesor fragments thereof, a cytokine, a receptor ligand and other suchligands. Representative examples of alpha-emitting isotopes includebismuth-213, bismuth-212, actinium-225, radium-223, lead-212,terbium-149, fermium-155 and astatine-211. The construct is highlyspecific with the activity of from about 0.1 mci/mg to about 50 mci/mgand administered in an effective dosage of from about 0.1 mg/m² to about10 mg/m². That is, the construct is administered in a dose adequate todeliver a minimum of 1 atom per cell. Representative examples ofnon-malignant cells that can be treated using this technique includevirus-infected cells, autoimmune cells, lymphoid cells, normal bonemarrow cells and abnormally proliferating normal cells. The individualmay have a disease such as neoplastic disease, viral infection,autoimmune disease, prostatic hypertrophy, coronary disease and othervascular occlusive disease.

The present invention is further directed to a method of killing a tumorby targeting antigens in the tumor vasculature of an individual in needof such of such treatment, comprising the step of: administering to saidindividual a pharmacologically effective dose of a construct comprisingan alpha-emitting isotope effective to inhibit the function of saidtumor vasculature. Representative examples of alpha-emitting constructsinclude antibodies or fragments thereof, cytokine, a receptor ligand andother such ligands. Preferably, the alpha-emitting construct is labeledby Bismuth-213, Bismuth-212, actinium-225, radium-223, lead-212,terbium-149, fermium-155 or Astatine-211. The construct is highlyspecific with the activity of from about 0.1 mci/mg to about 50 mci/mgand administered in an effective dosage of from about 0.1 mg/m² to about10 mg/m². That is, the construct is administered in a dose adequate todeliver a minimum of 1 atom per cell.

As used herein, “specific activity of an alpha-emitting construct”refers to refers to the number of radioactive atoms per ligand molecule.As used herein, “tumor control probability (TCP)” refers to theprobability that a tumor will be reduced to the size below which it cannot recur. As used herein, “remission duration” refers to the time aftertreatment before the tumor recurs. As used herein, “doubling time”refers to time it takes for a cancer cell or tumor to double in size.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

Example 1 Materials and Methods

Ac-225 is obtained from the European Institute for Transuranic Research,Karlsruhe, Germany or from the United States Department of Energy (OakRidge, Tenn. or Hanford, Wash.). Approximately 20-25 mCi of Ac-225residue was dried onto the inner surface of a glass ampoule that isattached with 1/32 inch diameter tubing to a 5 cm by 0.5 cmpolypropylene tube with barbed reducing fittings, sintered polyethyleneplugs, and some acid washed glass wool containing approximately 200 mgof dry AG MP-50 resin, 100-200 mesh, H⁺ form (BioRad Laboratories,Hercules, Calif.). The ampoule and column are disconnected, and theampoule fitted with two 3-way stopcocks. The residue in the glassampoule was exposed to 0.5 ml of 3 M Optima grade HCl (FisherScientific), added with a 3 ml syringe through one of the stopcocks. Theother end of the glass ampoule containing the Ac-225 acid solution wasvented to the atmosphere using a 0.22 mm filter (Corning) allowingrelease of any pressure increase due to heat or gas generation. The 3 Macid is allowed to contact the residue in the glass ampoule for 1 hourwith mild agitation to completely dissolve all of the Ac-225. After 1hour a second syringe containing 0.5 ml of metal free water wasattached, via the stopcock, and the acidic actinium chloride solutioncarefully diluted. The resulting 1.5 M acidic solution of actinium waswithdrawn into the syringe, the ampoule carefully disconnected and thesyringe with the Ac-225 solution attached to the ampoule.

The resin in the column was washed with 10 ml of 1.5 M Optima grade HCl,the resin was removed by backwashing and 100 mg was placed into a clean10 ml syringe in 2 ml of 1.5 M Optima grade HCl solution. Of theremaining 100 mg of washed resin, 50 mg was loaded back in the columnand a small piece of acid washed quartz glass wool added to hold theresin in place. This 50 mg section of resin serves as a catch plug tocapture any Ac-225 that might break through during routine elution. Thecolumn, the syringe with the Ac-225 solution, and the 10 ml syringe wereall attached via a 3-way plastic stopcock. The exit end of the columnwas attached to a 60 ml syringe that will be used to apply a negativepressure while filling the column.

Manipulation of the 3-way stopcock allows the Ac-225 solution to bepulled into the syringe containing the AG MP-50 resin slurry. ThisAc-225 solution and resin slurry were allowed to contact for 30 min.with occasional gentle agitation. After batch loading the Ac-225 ontothe resin support, the 3-way stopcock was again manipulated to load theAc-225/resin into the column. The apparatus was positioned so that thecolumn now stands vertically to allow the Ac-225/resin to flow downward.The resin was gently mixed prior to pulling it out of the 10 ml syringewith a slight negative pressure to pack the column. The stopcockposition originally used for the syringe with the Ac-225 solution wasnow attached to a clean syringe with 10 ml of 1.5 M Optima grade HClwashing solution. This wash was pulled into the 10 ml syringe where theresin was loaded, agitated slightly to rinse the syringe, and then usedto wash the generator column. The 60 ml syringe was used to pull thewash solution through the column. The column was disconnected from the3-way stopcock and a small plug of acid washed quartz glass wool isapplied to hold the resin in place. A layer of 50 mg of resin wasfurther added to serve as the second catch plug which is utilized whenthe column flow is reversed. A small piece of acid washed quartz glasswool was again added and the column completed by the addition of abarbed reducing fitting. The generator was then ready to use. It isrecommended to vertically position the generator when eluting so thatthe catch resin portion is always on top thus allowing any finesproduced to settle out and not clog the resin.

A 0.1M HCl/0.1M NaI buffer is prepared fresh each time and used to eluteBi-213 from the resin. The Bi-213 reaches secular equilibrium with theAc-225 after approx. 138-300 minutes (6.5 Bi-213 t_(1/2)'s). 2.5 ml of0.1M HCl/0.1M NaI elution buffer was required to elute 98% of therecoverable bismuth and 3.6 ml elutes all of the recoverable bismuth. Afreshly prepared 0.1M HCl/0.1M NaI solution is colorless, however afterpassage over the generator resin, it elutes as a faint yellow coloredsolution. Addition of 0.20 ml of 3M ammonium acetate was required tobuffer 3.6 ml of 0.1M HCl/0.1M NaI eluate to pH 4-5. A 150 mg/mlsolution of l-ascorbic acid was prepared in metal free water and batchreacted with Chelex-100 resin for twenty minutes. After twenty minutes,the l-ascorbic acid/Chelex 100 slurry is filtered through a 0.45 mmfilter and the metal free l-ascorbic acid eluate collected. A volume ofthis eluate is added to the buffered Bi-213 mixture to yield a finalconcentration of l-ascorbic acid equal to 5 mg/ml. The antibodyconstruct, CHX-A-DTPA-HuM195, was then added to the buffered Bi-213solution and incubated at room temperature for up to 20 minutes.Reactions taking greater than 4 minutes typically result in a greaterloss of Bi-213 product due to decay than allowing the reaction toproceed longer and increasing product yield. Following this incubation,0.020 ml of 10 mM EDTA solution was added to quench the reaction mixtureand chelate any free, reactive radiometal ions. Quenching was necessarybecause the reaction with the desired carrier may not proceedquantitatively (Bi-213 incorporation >98%) to desired product. Inaddition, a means of separating product from reactants and byproducts isalso needed. The radiolabelled antibody was purified from low molecularweight impurities rapidly by size exclusion chromatography.

Example 2 IgG

HuM195 (Protein Design Labs, Inc., Mountain View, Calif.) is arecombinant IgG1 mAb that was constructed by combining the CDR regionsof the murine M195 with human framework and constant regions. Both M195mAbs bind with high affinity to the CD33 antigen (22-26). The J591antibody, which is reactive with the Prostate Specific Membrane Antigen(PMSA) on prostate cancer cells, was the gift of Dr. Neil Bander at NewYork Hospital.

Example 3 Conjugation of Chelate to IgG

HuM195 was conjugated to 2-(p-SCN-Bz)-cyclohexyl-DTPA (CHX-A-DTPA), arecently developed backbone substituted derivative of DTPA24, using asingle vessel method (27) or conventional methods (28). The averagenumber of chelates per antibody ranged from 5 to 10.

Example 4 Radiolabelina

Bi-213: the radionuclide generator used for low activity level Bi-213production is described elsewhere (15, 29). The construction ofgenerators capable of producing 10-25 mCi of Bi-213 required severalmodifications. The generator was washed with 0.001 M HCl and then elutedwith 0.5 to 1 ml of 0.1 M HCl, 0.1 M NaI to elute Bi-213. Forradiolabeling of protein, the eluate was brought in the range of pH4-4.5 with 3 M ammonium acetate and immediately used as described forBi-206. Bi-212: Bismuth-212 was eluted from Ra-224/Bi-212-generator 14and HuM195 was labeled under similar conditions as described for Bi-213.

Example 5 Bi-213 Counting

Bi-213 radioactivity (photon energy 440 KeV, 28% abundance) wasquantified using a Squibb CRC-17 Radioisotope Calibrator at a fixedsetting that was standardized using a Canberra multi-channel pulseheight analyzer. A Packard Cobra gamma counter (340-540 KeV window) wasused to determine the relative number of Bi-213 counts in ITLC, ATLC,HPLC, Protein A, and cell samples. Other nuclides were counted usingstandard methods.

Example 6 Purification of Radiometal Chelate-Conjugated IgG

Radiolabeled CHX-A-DTPA-HuM195 constructs were purified either byhigh-performance liquid chromatography (HPLC) using a Bio-Sil-250 sizeexclusion column (600×7.5 mm) with 20 mM sodium acetate/150 mM sodiumchloride, pH 6.5 mobile phase or by using low pressure chromatographyemploying a 10 DG size exclusion column (Bio-Rad Laboratories, Inc.,Hercules, Calif.) with a 1% human serum albumin/0.9% sodium chloridemobile phase.

To determine the labeling efficiency and purity of the reaction mixtureand final product, a 5 ml sample was removed for instant thin layerchromatography (ITLC) (Gelman Science Inc., Ann Arbor, Mich.) 32. Theplates were developed with 10 mM EDTA. Under these conditions, mAbremains at the origin and free metal migrates with the solvent front.The strips were cut at rf=0.5 and counted in a gamma-counter.

Example 7 Conjugation of HuM195 to CHX-A-DTPA

HuM195 CHX-A-DTPA labeling efficiency with Bi-213 was typically over 90%at specific activities of up to 20 mCi/mg, but efficiency decreased indirect relationship to the specific activity desired. With specificactivities of 50 mCi/mg, 50-70% efficiencies were achieved. Thisreduction may have been due to the small amounts of antibody used toachieve the higher specific activities. The chelation reaction ran tonear completion (85%) in 6 min., but was allowed to continue for a full15-20 min. to optimize labeling. However, because of the short half-lifeof Bi-213, continuing the reaction beyond about 5 min. does not increasefinal yield of labeled product as product is lost through decay. Theselabeling efficiencies were comparable to those seen with In-111, Bi-206and Bi-212 using the CHX-A-DTPA-HuM195 construct.

Conjugation of HuM195 to CHX-A-DTPA resulted in the attachment of up to10 ligand molecules per antibody. High chelate to protein ratios did notsignificantly affect the immunoreactivity. The immunoreactivity of themetal-labeled CHX-A-DTPA-HuM195 varied from 80% to 95% and wasindependent of the specific activity. This is consistent with the aminoacid sequences in the CDR regions of the HuM195 (30).

Example 8 Immunoreactivity

The immunoreactivity of the bismuth labeled CHX-A-DTPA-HuM195 constructswas determined as described by incubating 2 ng of radiolabeled mAb in 30ml total volume with a 20- to 30-fold excess of antigen (10×106 or15×106 CD33 positive HL60 cells). These cells have approximately10,000-20,000 CD33 positive binding sites per cell and have the capacityto bind up to 90% of added HuM195). After incubation, the cells werecollected by centrifugation and unbound IgG was removed to a second setof the cells and reincubated with the same amount of excess antigen asin first incubation for 90 min. at 0° C. Under these conditions of largeantigen excess in a small volume, the reaction goes to near completionin 60 minutes. The percentage immunoreactivity was calculated as equalto (bound Bi-206-IgG to cells #1 plus cells #2)/(total bound plusunbound Bi-206-IgG) times 100. Specific binding in these radiobindingassays was confirmed by lack of binding of the radiolabeled mAb to CD33negative RAJI cells. To avoid nonspecific and Fc receptor binding, theassays were performed in the presence of 2% human serum.

A rapid, affinity thin-layer chromatography (ATLC) assay was implementedto measure the immunoreactivity of the short-lived Bi-213 constructs(31). The immunoreactivity was assessed as the percent radiolabeledconstruct bound to the portion of the paper strip containing the targetantigen which was prepared from extracts of AL67 cells (CD33transfectants).

Example 9 Modulation of Cell Surface Antibody-Antigen Complexes

Internalization of the cell surface antibody antigen-complex wasmeasured by incubating 0.5 mg/ml of radiolabeled mAb with 5×10⁵ cellsover time at 37° C. Cell pellets were washed twice in RPMI, and thensurface-bound (Bi-206)CHX-A-DTPA-HuM195 was stripped with 1 ml of 50 mMglycine/150 mM NaCl, pH 2.8, at 24° C. for 10 minutes. Totalcell-associated radioactivity and acid-resistant (internalized)radioactivity were determined. To avoid nonspecific and Fc binding, theassays were performed in the presence of 2% human serum.

Example 10 Cell Killing

The potency of (Bi-213)CHX-A-DTPA-HuM195 and (Bi-212)CHX-A-DTPA-HuM195for killing of leukemia cells was measured using 2×105 HL60 cells(CD33+) or RAJI cells (CD33−) in 100 ml in 96 well plates. Serialdilutions of bismuth labeled antibody were added to the wells to yieldfinal activity in the wells ranging from 0.02 to 20 mCi/ml. Theexperiments were done with different specific activities of the bismuthantibody (3 to 20 mCi/mg). The plates were incubated 24 h at 37° C. in5% CO2. After incubation, cell viability was determined by ³H-thymidineincorporation. To avoid nonspecific and Fc receptor binding duringincubation, the assays were performed in the presence of 2% human serum.

In addition, a bifunctional chelate, labeled to high specific activity,was conjugated with an alpha-emitting isotope. Internalization, highimmunoreactivity, tumor cells kill in vitro, the relationship betweencell kill and specific activity was demonstrated with a total of 5different monoclonal antibodies: HuM195 humanized anti-CD33 to myeloidleukemias, C2B8 chimeric anti-CD20 to lymphomas, J591 mouse anti-PSMA toprostate cancer, mouse SJ25C1 and mouse B4 anti-CD19 to B cell leukemiaand lymphoma. Thus, multiple examples with mouse, human, and chimericantibodies in several tumor antigen systems have been documented.

Example 11 Specific Cytotoxicity Killing of Large Tumor Clusters InVitro

FIG. 2 demonstrates, in a spheroid model, the possibility of killinglarger tumors by repeated dosing. As may be seen in the figure, a singledose has eliminated 5 to 6 layers of cells, leaving behind a previouslyunexposed “core” of cells that can then be targeted by a subsequentadministration. In contrast, spheroids of the same cell line, exposed toa Bi-213-labeled irrelevant antibody, continue to grow exponentially.These results are in contrast to the observations of Langmuir et al.(14), upon investigation of Bi-212-labeled antibody targeting of largespheroids, concluded that due to their short half-life and range of thealphas, alpha-emitters would not be effective against larger tumorsbecause of the time required for antibody penetration. FIG. 2illustrates that this will not be a limiting factor if repeated dosingis used.

Cell killing experiments with different specific activities of Bi-212 orBi-213 labeled CHX-A-DTPA-HuM195 showed dose- and specificactivity-dependent killing of CD33+ HL60 cells.(Bi-212)CHX-A-DTPA-HuM195 was at least 10 times more potent at killingCD33+ HL60 cells as compared to CD33 negative RAJI cells at 24 hours inin vitro assays (FIG. 3A). The potency against the specific target HL60cells depended directly on the specific activity, i.e., the number ofBi-212 per IgG molecule, of the labeled antibody with the highestspecific activities (30 mCi/mg) showing the highest potency. As specificactivity was decreased there was a loss of selective cell killing.Potency for killing HL60 cells at a specific activity of 0.2 mCi/mgapproached the potency for killing the RAJI control cells.

The dependency of selectivity on specific activity can be explained byexamining the number of CD33 target sites on each HL60 cell and thenumber of bismuth atoms labeled per HuM195 IgG molecule. At 0.2 mCi/mg,only about one IgG out of 100,000 contains a Bi-212. Because there areonly 10,000 CD33 sites per cell, it is unlikely that specific cellkilling can occur. Nonspecific cytotoxicity from alpha particleradiation in the media, or from antibody constructs nonspecificallybound to the cells then dominate the cytolytic activity. Thus, killingof HL60 cells at low specific activities of labeling approached that ofRAJI. Conversely, at specific activities of 20 mCi/mg, about one out of1000 HuM195 IgG molecules are labeled, thus allowing a mean of tenBi-212 atoms to be delivered to each HL60 cell at saturation. Therefore,at high specific activities, cytotoxicity should depend directly on thebinding characteristics of HuM195 for HL60 cells. The binding isothermdisplays exponential increase in binding from 10 to 1000 ng/ml.Nonspecific binding shows linear increases, beginning at higherconcentrations.

Similar specific killing of HL60 cells was observed for(Bi-213)CHX-A-DTPA-HuM195 (FIG. 3B). At specific activities of 8-10mCi/mg, potency against HL60 cells was about 10 fold higher than againstRAJI cells. As expected, (Bi-212)CHX-A-DTPA-HuM195 was slightly morepotent than (Bi-213)CHX-A-DTPA-HuM195; this is because at the samespecific activities, more Bi-212 would be conjugated per HuM195 IgG dueto its longer physical half-life. Therefore, for equivalent binding ofHuM195 to each cell, more alpha decays would be delivered byBi-212-constructs. As with Bi-212, the potency of cell kill dependeddirectly on the specific activity of bismuth atoms per IgG, as well ason the total dose added.

In order to determine the number of bismuth atoms necessary tospecifically kill HL60 cells, the cytotoxicity data were replotted as afunction of bismuth atoms per cell (FIG. 4). Data for killing at aspecific activity of 10 mCi/mg are shown. The HL60 cell viability was afunction of Bi-212 and Bi-213 atoms bound on the cell surface. Tocalculate the initial amount of bismuth atoms bound on the cell surface,specific activity and Scatchard analyses were used to estimate thepercentage of binding sites that are occupied with HuM195. The lines inFIG. 4 represent a best fit for the data points that are in the regionof specific killing. This is described by the function Y=111.24×e−ln2×0.419X for Bi-213; where Y is viability and X number of initialbismuth atoms. The function is Y=87.42×e−ln 2×0.429X for the Bi-212labeled antibody constructs. These data yield an LD50 dose of Bi-213 andBi-212 that is in the range of 2 to 2.5 initial atoms per cell.

To show that specific killing was not a unique property of the leukemiasystem, similar experiments with Bi-213 labeled J591 antibody (Liu,1997) and human prostate cancer cells (LnCaP cells) were conducted. Thesame dependence on specific activity in this system was observed (FIG.5).

Example 12 Treating Macroscopic Animal Tumors

To demonstrate that alpha-emitting ligands have an effect on macroscopictumors in vivo, an animal therapy study was conducted using nude miceinjected with 15 million LnCaP prostate tumor cells into their thighs.Tumors were allowed to grow until visible and were 3-5 mm in diameter.One group of mice was then treated with the Bi-213 labeled J591 antibodyspecific for prostate cancer cells and one group received a controlantibody at a slightly larger dose (Bi-213-HuM195). As a quantitativemeasure of antitumor activity, Prostate Specific Antigen (PSA) wasmeasured in the serum of mice before and after treatment. One week aftertreatment, mice given the control antibody showed a mean 26% rise in PSA(n=4) whereas mice treated with the specific J591 antibody showed only amean of 6% rise (n=4).

In a second experiment, 6 million prostate cancer cells were injectedinto mice. Mice were treated with a control Bi-213 radiolabeledantibody, no antibody, or Bi-213 labeled radioactive J591 prostatespecific antibody. By 28-31 days, both control groups had evidence ofcancer in 50% of the animals. In contrast, in the treatment group, itrequired 46 days before 50% of the animals showed tumors. Hence, thealpha-emitting prostate antibody was capable of slowing the growth of amacroscopic tumor. Other tumors that might be approached by such amethod include “benign” tumors such as “benign prostatic hypertrophy”caused by neoplastic, but not malignant, overgrowth of the prostate.This condition afflicts millions of men worldwide.

Example 13 Cell Killing In Vivo by Bi-213 J591

A single course of the Bi-213 J591 drug was administered to the LNCaPmouse model in four daily doses. The results show that median tumor-freesurvival of LNCaP xenografted mice was improved (p<0.0031) relative tomice treated with Bi-213-HuM195 or untreated controls (FIG. 6). Themedian tumor-free survival times were 31 days (n=4, untreated animals),33 days (n=6, Bi-213-HuM195-treated animals), and 54 days (n=6,Bi-213-J591-treated animals).

PSA levels were also evaluated in tumor-bearing mice. It shows that themice responded to the treatment (Table 3). Mean PSA values, 51 daysafter treatment, were 104 ng/ml±54 ng/ml (n=4, untreated animals), 66ng/ml±16 ng/ml (n=6, Bi-213-HuM195-treated animals), and 28 ng/ml±22(n=6, Bi-213-J591-treated animals). The reduction of PSA levels in micetreated with Bi-213-J591 relative to mice treated with Bi-213-HuM195 anduntreated control animals was significant with p<0.007 and p<0.0136,respectively. In another similar experiment where unlabeled J591 wasalso examined as an additional control, the mean PSA values 30 daysafter treatment were 31 ng/ml±20 ng/ml (n=5, untreated animals), 36ng/ml±38 ng/ml (n=5, 0.02 mg J591-treated animals), 26 ng/ml±21 ng/ml(n=10, Bi-213-HuM195-treated animals), and 12 ng/ml±8 ng/ml (n=12,Bi-213-J591-treated animals) (FIG. 7).

In this experiment, animals received either one single Bi-213 drugadministration or four consecutive daily administrations of a smallerdose of drug. There were no statistically significant differences in theresponses, i.e., measured PSA levels, observed between the 1× daily andthe 4× daily treatment regimens for the Bi-213-J591 and theBi-213-HuM195 treatments, respectively, nor between the unlabeled J591and untreated controls. Reduction of PSA levels, however, in all mice(n=12) treated with Bi-213-J591 (1× daily and the 4× daily treatmentregimens pooled) relative to all mice (n=10) treated with Bi-213-HuM195(1× daily and the 4× daily treatment regimens pooled) and all controlanimals (groups untreated and treated with unlabeled J591 pooled; n=10)was significant with p<0.0443 and p<0.0192, respectively.

TABLE 3 P for the observed PSA values between the Bi-213-J591-treatedmice and the Bi-213-HuM195-treated mice and controls Bi-213-HuM195Controls Day 30 Bi-213-J591 (n = 12) p < 0.0443 (n = 10) p < 0.0192 (n =10) Day 51 Bi-213-J591 (n = 6) p < 0.007 (n = 6) p < 0.0136 (n = 4)

Example 14 Killing Larger Tumors by Alpha Particle Killing ofVasculature

Because the ligand carrying the alpha-emitting isotope is unlikely todiffuse into a large tumor during the period in which the isotope isstill radioactive, due to the short half life of the isotopes, analternative method for killing with these constructs is to kill withoutthe need for diffusion. For example, it is possible to rapidly deliverwithin a few minutes the alpha-emitting ligand to the blood in thewell-vascularized tissues and organs. If one can target the tumorvasculature itself, rather than the tumor cells, it will be possible tokill the tumors selectively by this method. Such a method has beendemonstrated for native or chemotherapy conjugated ligand or antibody(32). No suggestion of using an alpha particle emitting construct wasenvisioned in this prior art. However, based on the data contained here,it can be inferred that such an approach would be successful as well.One such ligand capable of targeting vasculature is the J591 antibody(33). This antibody targets the PSM antigen found selectively on tumorvasculature as well as on prostate cancer cells.

In contrast to the teachings of the prior art, solid tumors can betreated by alpha conjugated ligands and it is possible to kill largertumors using short-lived alpha-emitting ligands. The present inventiondemonstrated in an in vitro model using tumor clusters “spheroids”containing many thousands of cells that it is possible to firstselectively kill the outer layers of a tumor (2-4 cells thick) and thusexpose the next inner layers for killing. In this way, by repeated dosesof drug, separated in time to allow the death of the outer layers, it ispossible to kill larger tumors. This method can be likened to “skinningan onion” and was not obvious until it was demonstrated in vitro and invivo herein. Finally, it was shown to be true in a live animal modelbearing macroscopic tumors.

Cell killing experiments showed specific cell killing with the(Bi-213)CHX-A-DTPA-HuM195, (Bi-212)CHX-A-DTPA-HuM195 and(Bi-213)CHX-A-DTPA-J591 constructs which was dependent on both dose andspecific activity of labeling. Both bismuth isotopes showedapproximately 50% killing when two bismuth atoms were initially boundonto the target cell surface. Because there are about 10,000 CD33 sitesper cell, this implies that only 1 mAb in several thousand will need tobe labeled in order to get high levels of cell kill. Because of therapidly diminishing solid angle occupied by the target cells relative tothe IgG in solution, the possibility that alpha-particles that areemitted from starting points beyond the target cell surface may hit thecell nucleus becomes negligible at short distances away from the cell.Similarly, the most efficient cell killing will occur from thoseemissions that occur from internalized bismuth. An emission from asurface bound IgG may also pass harmlessly away from the cell. Becauseapproximately 50% of radiolabeled CHX-A-DTPA-HuM195 is internalized intocell in 60 min., these data suggest that it is likely that one alphaemission from one atom within the cell is capable of killing that cell.When the initial number of bismuth atoms bound per cell is 2 to 2.5 andthe average internalization time is 60 min., there are approximately 60%of cells in which no bismuth atom is inside, based on the Poissondistribution and the probability that the bismuth atom is internalized.At high specific activities, 50% killing of HL60 cells at 24 hr wasobserved with the alpha-emitting constructs at IgG concentrations of3.3-25 ng/ml (20-160 pM). A conversion to actual bismuth-labeled IgGshows that only 5-10 pg/ml (30-60 fM) of the radioimmunoconjugate arerequired at the 50% effective dose.

An important characteristic of the curves describing the cell kill byalpha emitters is the marked dependence on specific activity of theradioconjugate. This was shown with 3 experiments, using 2 differentalpha-emitting isotopes and both a leukemia and a solid tumor. Becausekilling requires specific delivery of the bismuth to or into the cell,as the number of bismuth atoms per HuM195 falls to levels near 1 bismuthper 10,000 IgG molecules, the ability to kill targets approaches thatseen with a non-specific target cell. In this instance, unlabeled HuM195competes for sites with the bismuth labeled CHX-A-DTPA-HuM195. Thiseffect is quite pronounced in this system because of the small number oftarget binding sites (about 1-4×10⁴) on HL60 cells, and LnCaP cellsrespectively.

Example 15 Demonstration of the Clinical Usefulness of the High SpecificActivity Ligand for Treating a Human Cancer in Patients

A clinical experiment or protocol describing one possible use of analpha-emitting targeted construct follows. This experiment describes theuse of the HuM195 IgG1 to target Bismuth-213 to leukemia cells anddemonstrates that the constructs are stable, will deliver the isotope tothe cells in a human, and will kill leukemia cells without apparenttoxicity to non target tissues. Such a scheme might be used with anotheralpha emitter, such as bismuth-212 attached stably to this or anotherligand, such as an antibody or fragment, cytokine, or receptor ligand,each of which is capable of specific and high affinity binding to atarget cell or tissue. Moreover, such ligands might be used also toselectively kill nonmalignant targets such as lymphoid cells involved ina pathological process such as inflammation or autoimmunity, or normalbone marrow cells to enable a bone marrow transplant or transplant ofanother organ or tissue, or overgrown normal cells that need to bekilled because they are involved in a pathological process such ascoronary artery disease or other vascular occlusive diseases. Theobjectives of the clinical protocol are to determine the safety andtoxicity of 213-Bi-labeled HuM195 in patients with relapsed orrefractory myeloid malignancies, to determine the pharmacology anddosimetry of 213-Bi-HuM195 and to study the biological effects of213-Bi-HuM195, including the ability to elicit human antihuman antibody(HAHA) responses and antileukemic responses.

Acute myelogenous leukemia (AML) is the predominant type of acuteleukemia in adults. While most patients are able to achieve a completeremission with chemotherapy consisting of cytosine arabinoside and ananthracycline, prolonged disease-free survival is less than 20%.Reinduction attempts will produce second remissions in only 20-25% ofpatients, frequently lasting less than 6 months. Less than 5% ofrelapsed patients will survive one year.

Chronic myeloid leukemia (CML) is a biphasic disorder of earlyhematopoietic progenitors. The chronic phase (with a median duration of4 years) is associated with marked elevations of mature and maturingleukocytes and leads invariably to a blastic phase resembling acuteleukemia. Treatment with “a-interferon has been shown to eradicateevidence of the Philadelphia chromosome by cytogenetic analysis in aminority of patients. Treatment with conventional chemotherapy, however,has had no impact on the natural history of this disease. Allogeneicbone marrow transplantation is the only potentially curative option forthese patients. Since patients in accelerated or blastic phases of CMLgenerally do not benefit from transplantation, efforts have been made totransplant these patients during the early chronic phase of theirdisease.

Classified as a myelodysplastic syndrome, chronic myelomonocyticleukemia (CMMOL) is defined by the presence of a monocyte count ofgreater than 1 H 109/L, monocytosis of the bone marrow, anemia, andthrombocytopenia. Survival ranges from several weeks to years, with amedian survival of 30 to 41 months. Treatment is mostly palliative;hydroxyurea can be used to control high peripheral blood leukocytecounts.

The CD33 antigen is distinctive among hematopoietic antigens in itsrestricted distribution, and M195 (anti-CD33) is effective in targetingleukemia cells in vitro. M195 is a monoclonal IgG2a antibody derivedfrom a mouse immunized with live human leukemic myeloblasts. Bindingspecificity of M195 is restricted to myeloid and monocytic leukemia celllines and a fraction of mature adherent monocytes. Approximately 10,000antibody-binding sites per cell are expressed on myeloid or monocyticleukemia cell lines and 5,000 sites on mature monocytes.

M195 shows targeting to leukemia cells in humans. Ten patients withmyeloid leukemias were treated in a phase I trial with escalating dosesof mouse M195. M195 was trace-labeled with ¹³¹I to allow detailedpharmacokinetic and dosimetric studies by serial sampling of blood andbone marrow and whole body gamma-camera imaging. Total doses up to 76 mg(40 mg/m²) were administered safely without any immediate adverseeffects. Adsorption of M195 onto leukemic cells in vivo was demonstratedby biopsy, pharmacology, flow cytometry, and imaging. Saturation ofavailable binding sites occurred at doses of greater than 5 mg/m2. Theentire bone marrow was imaged specifically and clearly beginning withinseveral hours after injection. M195 was rapidly internalized afterbinding to target cells. An estimated dose of up to 34 rad/mCi wasdelivered to the marrow, indicating that whole bone marrow ablativedoses of ¹³¹I could be carried by M195.

Humanized M195 (HuM195) is a fully humanized M195 construct that hasimproved biochemical and immunological activities.Complementarity-determining region (CDR)-grafted humanized M195,retaining only the CDRs and other sterically important amino acids frommouse M195 were constructed using human IgG1 frameworks. Sp2/0 mousemyeloma cell lines secreting humanized M195 were grown in vitro and theantibodies were purified on PA-Sepharose by affinity chromatographyusing sequential pH step elutions. Purity was determined onSDS-polyacrylamide gels stained with Coomassie brilliant blue. TheHuM195 construct maintained binding specificity confirmed against apanel of CD33+ and CD33− cell lines by radioimmunoassays.

HuM195 shows specific targeting of leukemia without immunogenicity invivo. Toxicity, pharmacology, dosimetry, development of human antihumanantibody (HAHA) responses, and antileukemic effects of HuM195 werestudied in patients with relapsed or refractory myeloid leukemias.Thirteen patients were treated on a twice weekly schedule for 3 weeks at4 dose levels ranging from 0.5 to 10 mg/m2. Alpha emitters have now beenconjugated via a bifunctional chelate (CHX-A-DTPA) to HuM195 with highefficiency (>90%) and high specific activities (up to 20 mCi/mg)

Example 16 Antibody Production and Labeling

HuM195 is produced by Protein Design Labs, Inc. (Mountain View, Calif.).Sp2/0 hybridoma cell lines secreting HuM195 are grown in serum-freemedium. HuM195 is purified from concentrated supernatants by affinitychromatography followed by additional purification steps.HuM195-CHX-A-DTPA is prepared on contract by TSI Washington (Rockville,Md.). HuM195-CHX-A-DTPA is supplied as a solution at 10.6 mg/ml andstored at −70° C.

Clinical grade 213-Bi generators capable of producing 25-50 mCi areprepared at Sloan-Kettering. Actinium is supplied dried onto a glassampule from the Transuranium Elements Institute in Karlsruhe, Germany.213-Bi is eluted from the generator and chelated to HuM195-CHX-A-DTPA,followed by separation of Bi-213-HuM195 by size exclusionchromatography. Sensors for OD280 and gamma emissions are used todetermine yield and specific activity. Unlabeled HuM195 may be added toadjust the dose as necessary. This will be performed at Sloan-KetteringInstitute immediately prior to injection into patients. Bi-213-HuM195 isdiluted in normal saline with 1% human serum albumin (HSA) to a totalvolume of 10 ml for injection. Bi-213-HuM195-CHX-A-DTPA is manufacturedand tested according to an FDA approved IND.

The patient eligibility requirements are that (1) patients must havepathologically confirmed diagnosis of AML that is in relapse orrefractory to at least 2 courses of standard induction chemotherapy),accelerated or myeloblastic phase of CML, or CMMOL; (2) greater than 25%of bone marrow blasts must be CD33 positive; (3) patients must have alife expectancy of at least 6 weeks and a Karnofsky performance statusof >60%; (4) patients must not have received chemotherapy orradiotherapy for at least 3 weeks prior to treatment, except forhydroxyurea which must be discontinued 2 days prior to treatment.Patients must have recovered from the effects of previous treatment andshow clear signs of active leukemia. Patients must not have rapidlyaccelerating blast counts or clinically unstable disease; (5) Patientsmust have a serum creatinine <1.5 times the upper limit of normal,bilirubin #1.0 mg/dl, and alkaline phosphatase and SGOT #2.5 times theupper limit of normal which represent grade 0 or 1 toxicities by the NCIcommon toxicity criteria; and (6) Patients must sign informed consent.

Patients are treated on either an outpatient or inpatient basis. Giventhe short-half life of Bi-213, radiation exposure to hospital staff isminimal and radiation isolation for patients is not required. Patientsare monitored by a Radiation Safety Officer and instructed in the properdisposal of waste. Additionally, patients are discharged for two tothree hours after infusion, by which time any remaining gamma radiationwill be trivial. Patients will receive Bi-213-HuM195 by IV push, individed doses, 1-4 times daily, 3-4 hours apart. The following doseescalation scheme is employed: Dose Level 1 (0.28 mCi/kg); Dose Level 2(0.42 mCi/kg); Dose Level 3 (0.56 mCi/kg); Dose Level 4 (0.7 mCi/kg) andso on with further escalations as needed.

Vital signs, e.g., pulse, blood pressure, respiratory rate, temperature,will be monitored and recorded before treatment, 30 and 60 minutesfollowing infusion, and hourly thereafter for 4 hours. The followingtests will be done: CBC, differential, platelet count: Twice dailyduring treatment, then weekly Q 4, then monthly Q 3. Electrolytes, BUN,Creatinine, Biochemistry profile: Daily during treatment, then weekly Q4, then monthly Q 3. Human antihuman antibodies: Before treatment andmonthly thereafter Q 4. Gamma camera imaging: Continuously for 60 min.after injection of the first and last dose and again at 90 min. afterinjection of first and last dose. Bone marrow and biopsy, includingimmunophenotyping: 7-10 days and 4 weeks after treatment.Pharmacokinetics: 5, 10, 15, 30, 45, 60, 90, 120, and 180 minutes afterfirst and last dose.

Twelve patients have entered on four dose levels. More than 50 doses ofthe drug were synthesized according to the specifications and injectedinto the patients. Doses could be prepared from the generator at leastevery 3-4 hours. The generator provided drug that met specifications forat least nine days allowing the treatment of patients. Real timepharmacokinetics were assessed by gamma imaging and serial blood work.The drug targeted first to the sites of leukemia and monocyte/macrophagecells in the liver and spleen. The bone marrow was targeted next. Overtime, with succeeding injections, uptake into the liver decreased by 50%as sites were saturated, and uptake in the bone marrow increased by100%, as more drug became available.

The estimated radiation doses in REM to the whole body, kidneys or othernon-target organs were 0.03; to the blood, 125; to the liver 600; to thespleen 1400; l and to the red marrow, 1100. Target to non-target doseratios were therefore 25,000-50,000 to one. There was no acute toxicityin any patient. There was no extramedullary toxicity seen in anypatient. In most patients, peripheral blood cell counts of leukemiablasts and white cells began to fall within 48 hours after treatment andwere reduced by up to 90%. Counts returned within two weeks. The bonemarrow at one week showed reduced cellularity and reduced leukemia blastpercentages in the majority of patients (up to 70% reduced).

Example 17 Ac-225 Labeled Constructs

Ac-225 labeled constructs are evaluated for stability in vitro at 37° C.using a Protein A Bead assay that evaluates free Ac-225 and Ig-boundAc-225 (Nikula et al., 1999). Radionuclide detection is carried-outusing gas ionization detection (GID) or pulse height multi-channelanalysis (MCA) with a high purity Ge detector (HPGe).

The potency and specificity of the relevant Ac-225 labeled constructsvs. labeled control constructs in killing single cells and multicellularspheroids are evaluated in vitro as a function of specific activity andactivity concentration. A ³H-thymidine incorporation assay is used toascertain the number of cells surviving exposure to targeted Ac-225. Theability, and where applicable, the rate of internalization, of Ac-225labeled IgG constructs into target cell lines is investigated as abovedescribed for the B1-213 labeled HuM195 construct.

The retention of Ac-225 in individual cells and multicellular spheroidsas a function of time is evaluated by exposing an excess of Ac-225labeled IgG construct to cell antigens, incubating for an appropriatetime, washing, and resuspending in fresh media. The reactivity remainingwith the cells and the activity in the supernatant and wash is thenquantified. The exposed cells and spheroids are held for 10 days whichis one Ac-225 half-life and each day the cells are centrifuged and afixed volume of supernatant and a fixed volume of concentrated cellssampled for the relative levels of Ac-225 and the radionuclidecomposition of the respective components. Radionuclide quantitation andcharacterization is carried-out as previously described.

A variety of novel chelating agents that may be potentially more stablein vivo than [Ac-225]CHX-A-DTPA moiety will be utilized. Initially, thechelation capability is investigated to determine the relative kineticsand thermodynamics of actinium chelation. The rate of chelateincorporation of Ac-225 is assessed by instant thin layer chromatographytechniques using silica gel impregnated paper and a basic pH, aqueousmobile phase. Thermodynamic stability is assessed on a relative basisvs. EDTA challenge and incubation in serum or media at 37° C. forseveral days. Chelating agents are evaluated in this fashion to findthose capable of rapid on-reaction, high kd, and suitable stability forin vivo utilization. Candidate chelating agents are further developed bypreparing constructs with the mAb's studied.

Example 18 Preliminary Results on Ac-225 Constructs

Several Ac-225 labeling conditions have been evaluated and conditionsfor rapid labeling have been determined. Yields of [Ac-225]-CHX-AHuM195are 30-40% after 10 minutes at room temperature. Stability of CHX-A-DTPAchelating moiety in vitro was examined at 1, 3, 24 and 96 hours, and thepercentage of Ac-225 bound to HuM195 was 100±0, 100±0, 84±2, and 44±13,respectively for duplicate measurements.

HL60 vs. Raji cell killing with [Ac-225]HuM195 at specific activities of0.12 and 0.0012 Ci/g demonstrated specific, potent cell killing in a 48hours exposure assay. The LD95 was approximately 0.5, 50, 50, 50 nCi/mlfor the HL60 treated with 0.12 Ci/g, HL60 (0.0012 Ci/g), Raji (0.12Ci/g), Raji (0.0012 Ci/g), respectively. In comparison, the[Bi-213]HuM195 construct at specific activities of 8 Ci/g had LD95values of approximately 3 and 10 μCi/ml for HL60 and Raji, respectively.This represents a 67-fold decrease in the Ac-225 specific activity vs.Bi-213 and 6000-fold decrease in the amount of activity necessary tokill 95% of the cells.

AL67 and LNCaP.FGC spheroid cell killing experiments at 0.02 to 8 mCi/mlhave been carried out and specific spheroid cell kill demonstrated usingthe HuM195 and J591 antibody constructs, respectively.

Example 19 LNCaP Tumor Model in Mice

A human prostate cancer tumor model was established in male athymic nudemice by intramuscular xenograft of 5E6 LNCaP tumor cells in the hind legon day 0. Tumor growth in vivo was assessed at several early time pointsby sacrificing mice and examining the morphology, size, vascularization,and encapsulation of the tumor cells in the leg histologically. SerumPSA levels in xenografted mice were determined on day 10-12post-xenograft. Mice were treated by intraperitoneal injection of200-300 nCi Ac-225 labeled anti-PSMA IgG (J591) on day 12-15post-xenograft. Animals were monitored for survival and PSA followingtreatment. Controls consisted of irrelevant Ac-225 labeled IgG (B4),doses of unlabeled anti-PSMA IgG, and untreated growth controls werealso performed.

Example 20 Ac-225 Therapy Against Solid Tumors

Histopathological evaluation of the LNCaP xenografted mice indicatedthat tumors were vascularized at day 10, and the encapsulated noduleswere found 1 mm×2 mm in size. Serum PSA levels were ranged from 2-5ng/ml on day 10-12 post-xenograft. FIG. 8 demonstrates composite ofAc-225 RIT I, II, III experiments on LNCaP xenografted mice. It showsthat alpha-emitting isotopes attached to monoclonal antibodies targetingthe tumors were capable of significantly prolonging the lives of orincreasing the curing effect on animals with solid tumors. It is alsoshown that isotope on irrelevant antibody (B4) were not effective, norwere non-radioactive antibodies.

The following references were cited herein.

-   1. O'Donoghue et al., (1995) J, Nucl. Med., 36:1902-1909.-   2. Willinset al., (1994) J. Nucl. Med., 35:123 P (abstract).-   3. Willins et al., (1995) J. Nucl. Med., 36:100-103.-   4. Sgouros, G. (1995) J. Nucl. Med., 36:1910-1912.-   5. Gerlowski et al., (1986) Microvasc. Res., 31:288-305.-   6. Dvorak et al., (1988) Am. J. Path., 133:95-109-   7. Jain et al., (1988) Cancer Res., 48:7022-7032.-   8. Clauss et al., (1990) Cancer Res., 50:3487-3492.-   9. Fujimori, et al., (1990) J. Nucl. Med., 31:1191-1198.-   10. Sgouros, et al., (1989) J. Nucl. Med., 30:777 (abstract).-   11. Sgouros, G. (1992) J. Nucl. Med., 33:2167-2179.-   12. Saga, et al., (1995) Proc. Natl. Acad. Sci., U.S.A., 92,    8999-9003.-   13. Riethmulleret al., (1994) Lancet, 343, 1177-1183.-   14. Langmuir et al., (1990) J. Nucl. Med. 31:1527-1533.-   15. Geerlings et al., (1993) Nucl. Med. Comm. 14:121-125.-   16. Simonson et al., (1990) Cancer Res. 50:985s-988s.-   17. Huneke et al., (1992) Cancer Res. 52:5818-5820.-   18. Macklis et al., (1992) Radiat. Res. 130: 220-226.-   19. Kozak et al., (1986) Proc. Natl. Acad. Sci. USA 83:474-478.-   20. Scheinberg et al., (1982) Science, 215:1511-1513.-   21. Steel, G. G. (1989) Cell Proliferation Kinetics In Tumours. In:    Steel, et al., (eds.), The Biological Basis of Radiotherapy, 2nd    Ed., Elsevier, Amsterdam.-   22. Scheinberg et al., (1989) Leukemia 3:440-445.-   23. Caron et al., (1992) Cancer Res. 52:6761-6767.-   24. Caron et al., (1994) Blood 83:1760-1768.-   25. Schwartz et al., (1993) J. Clin. Oncol. 11:294-303.-   26. Co et al., (1992) J. Immunol. 148:1149-1154.-   27. Nikula et al., (1995a) Nucl. Med. Biol. 22:387-390.-   28. Mirzadeh et al., (1990) Bioconjug. Chem. 1:59-65.-   29. Kaspersen et al., (1995) Nucl. Med. Communications 16:468-476.-   30. Nikula et al., (1995b) Molec. Immun. 32:865-872.-   31. Zamora et al., (1994) BioTechniques, 16:306-311.-   32. Arap et al., (1998) Science, 279:377-380.-   33. Lui et al, (1997) Ca. Research, 97:3629-3634.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1-20. (canceled)
 21. A method for treating a micrometastatic cancer In apatient, the method comprising administering to the patient a conjugatecomprising bismuth-213 or actinium-225 and an antibody specific for acancer cell antigen.
 22. A method for treating a non-malignant celldisease in a patient, the method comprising administering to the patienta conjugate comprising bismuth-213 or actinium-225 and an antibodyspecific for a non-malignant cell antigen.
 23. The method of claim 21 or22, wherein the conjugate is administered at a dose from about 0.1 mg/m²to about 10 mg/m².
 24. The method of claim 21 or 22, wherein thespecific activity of the bismuth-213 conjugate is about 20 mCi/mg toabout 30 mCi/mg.
 25. The method of claim 21 or 22, wherein the conjugateis administered intravenously.
 26. The method of claim 21 or 22, whereinthe specific activity of the actinium-225 conjugate is about 0.1 mCi/mgto about 0.15 mCi/mg.
 27. The method of claim 21, wherein themicrometastatic cancer is in a bone marrow of the patient.
 28. Themethod of claim 21, wherein the micrometastatic cancer is a leukemia.29. The method of claim 28, wherein the leukemia is an acute myelogenousleukemia, a chronic myeloid leukemia or a chronic myelomonocyticleukemia.
 30. The method of claim 21, wherein the micrometastatic canceris a peritoneal metastases.
 31. The method of claim 21, wherein themicrometastatic cancer Is a cancerous meningitis.
 32. The method ofclaim 31, wherein the cancerous meningitis Is a cancerous meningitis ina cerebrospinal fluid of the patient.
 33. The method of any of claim 21,27, 28, 30 or 31, wherein the micrometastatic cancer comprises a solidtumor greater than 1 mm in diameter.
 34. The method of any of claim 21,27, 28, 30 or 31, wherein the micrometastatic cancer comprises a solidtumor less than 1 mm in diameter.
 35. The method of claim 22, whereinthe non-malignant cell disease is selected from the group consisting aneoplastic disease, an infection, an autoimmune disease, a prostatichypertrophy, a coronary disease and a vascular occlusive disease. 36.The method of claim 35, wherein the infection is a viral infection. 37.The method of claim 22, wherein the non-malignant cell is selected fromthe group consisting of an infectious cell, an infected cell, anautoimmune cell, a lymphoid cell, a normal bone marrow cell and anabnormally proliferating normal cell.
 38. The method of claim 21 or 22,wherein the antigen Is a tumor vasculature antigen.
 39. The method ofclaim 21 or 22, wherein the antigen is PMSA.
 40. The method of claim 21or 22, wherein the antigen is CD33.
 41. The method of claim 21 or 22,wherein the antigen is GD3.
 42. The method of claim 21 or 22, whereinthe antigen is CD20.
 43. The method of claim 21 or 22, wherein theantigen is CD19.